Morphological mutants of filamentous fungi

ABSTRACT

The present invention relates to methods of obtaining a mutant cell from a filamentous fungal parent cell, comprising: (a) obtaining mutant cells of the parent cell; (b) identifying the mutant cell which exhibits a more restricted colonial phenotype and/or a more extensive hyphal branching than the parent cell; and (c) identifying the mutant cell which has an improved property for production of a heterologous polypeptide than the parent cell, when the mutant and parent cells are cultured under the same conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.08/816,239 filed Mar. 13, 1997, now U.S. Pat. No. 6,066,493, which is acontinuation-in-part of U.S. application Ser. No. 08/726,114 filed Oct.4,1996, now abandoned, which claims priority from U.S. provisionalapplication Ser. No. 60/010,238 filed Jan. 19, 1996, which applicationsare fully incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel fungi having improved capacityfor secretion of recombinant polypeptides, and a method for improvingsuch secretion.

2. Description of the Related Art

Filamentous fungi have become established as widely used host cellsystems for the production of recombinant polypeptides. In many cases,however, fungi which have the desirable traits of ease oftransformability and heterologous polypeptide expression do notnecessarily have the most desirable characteristics for successfulfermentation. For example, growth morphology during fermentation may notbe optimal, since many cultures become quite viscous as biomassincreases. Increased viscosity limits the ability to mix and aerate thefermentation culture, leading to oxygen and nutrient starvation of themycelia, which in turn become inviable and unproductive limiting theyield of the polypeptide of interest. On the other hand, filamentousfungal strains showing good fermentation morphology are not necessarilythe best production strains in terms of quantity of enzyme produced.Therefore, for commercial purposes, there is a need for filamentousfungal hosts which combine the capacity for expression of commercialquantities of recombinant polyp eptide with satisfactory growthcharacteristics, such as rapid growth and low viscosity, therebyenhancing productivity during fermentation.

Screening of large numbers of mutants for improved fermentationmorphology is quite difficult, and morphological mutant strains haveoften been isolated based on unusual colony morphology on solid medium.Traditionally, morphological mutants have been isolated in transformedstrains that contain multiple copies of a heterologous gene. The mutantsare then analyzed for fermentation growth characteristics andheterologous gene expression. Although this method may be useful inidentifying improved expression strains, the relationship between anyparticular fungal growth morphology and a strain's ability to produce alarge quantity of secreted polypeptide has yet to be established.Morphological mutants are also occasionally recovered in polypeptideexpression improvement screens, following mutagenesis of a transformedstrain, but again, the study of these strains has not led to anysignificant insight into the control of morphology. In addition,morphologically “improved” strains of parental strains containingheterologous gene expression cassettes are not suitable as generalexpression hosts since they cannot be used for the exclusive expressionof other heterologous polypeptides.

It is an object of the present invention to provide methods forproducing and identifying useful morphological mutants for heterologouspolypeptide production.

SUMMARY OF THE INVENTION

The present invention relates to methods of obtaining a mutant cell froma filamentous fungal parent cell, comprising: (a) obtaining mutant cellsof the parent cell; (b) identifying the mutant cell which exhibits amore restricted colonial phenotype and/or more extensive hyphalbranching than the parent cell; and (c) identifying the mutant cellwhich has an improved property for production of a heterologouspolypeptide than the parent cell, when the mutant and parent cells arecultured under the same conditions.

The invention also relates to mutant filamentous fungal cells producedby the methods of the present invention.

The present invention also relates to methods for producing aheterologous polypeptide, comprising: (a) culturing a mutant cell of thepresent invention which comprises a nucleic acid sequence encoding theheterologous polypeptide; and (b) recovering the heterologouspolypeptide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a restriction map of pJeRS23.

FIG. 2 shows the colony growth of control strain HowB425 and colonialmutant JeRS316 on PDA+uridine solid medium.

FIG. 3 shows a graphic illustration of the distribution of lipaseexpression in HowB425 (control) and JeRS316 (mutant) transformants.

FIG. 4 shows a graphic illustration of the distribution of lipaseexpression in HowB425 (control) and JeRS317 (mutant) transformants.

FIG. 5 shows a comparison of the heterologous lipase productive phase ofthe fermentation of control strain vs. mutant strain.

FIG. 6 shows a restriction map of pCaHj418.

FIG. 7 shows a restriction map of pDM148.

FIG. 8 shows a restriction map of pDM149.

FIG. 9 shows a restriction map of pJaL154.

FIG. 10 shows a restriction map of pMT1612.

FIG. 11 shows a restriction map of pJRoy30.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of obtaining a mutant cell froma filamentous fungal parent cell, comprising: (a) obtaining mutant cellsof the parent cell; (b) identifying the mutant cell which exhibits amore restricted colonial phenotype and/or more extensive hyphalbranching than the parent cell; and (c) identifying the mutant cellwhich has an improved property for production of a heterologouspolypeptide than the parent cell, when the mutant and parent cells arecultured under the same conditions.

The parent cell may be mutagenized by methods known in the art. Forexample, mutagenesis of the parent cell can be achieved by irradiation,e.g., UV, X-ray, or gamma radiation of the parent cell. Furthermore,mutagenesis can be obtained by treatment with chemical mutagens, e.g.,nitrous acid, nitrosamines, methyl nitrosoguanidine, and base analoguessuch as 5-bromouracil. Most conveniently, the mutagen is applied tospores of the parent strain, and the surviving spores are plated out forgrowth on a solid medium. It will also be understood that mutants canalso be naturally occurring variants in a population in the absence of aspecific mutagenesis procedure, either by selection, screening, or acombination of selection and screening. See, for example, Wiebe et al.,1992, Mycological Research 96: 555–562 and Wiebe et al., 1991,Mycological Research 95: 1284–1288 for isolating morphological mutantsof Fusarium strain A3/5. Therefore, for purposes of the presentinvention, the term “mutants” also encompasses naturally occurringvariants or mutants without deliberate application of mutagens, i.e.,spontaneous mutants.

The filamentous fungal parent cell may be any filamentous fungal cell.Filamentous fungi include all filamentous forms of the subdivisionEumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworthand Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK). The filamentous fungiare characterized by a vegetative mycelium composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic.

In the present invention, the filamentous fungal parent cell may be acell of a species of, but not limited to, Acremonium, Aspergillus,Fusarium, Humicola, Myceliophthora, Mucor, Neurospora, Penicillium,Thielavia, Tolypocladium, and Trichoderma or teleomorphs or synonymsthereof. Known teleomorphs of Aspergillus include Eurotium, Neosartorya,and Emericella. Strains of Aspergillus and teleomorphs thereof arereadily accessible to the public in a number of culture collections,such as the American Type Culture Collection (ATCC), Deutsche Sammlungvon Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau VoorSchimmelcultures (CBS), and Agricultural Research Service Patent CultureCollection, Northern Regional Research Center (NRRL). Known teleomorphsof Fusarium of the section Discolor include Gibberella gordonii,Gibberella cyanea, Gubberella pulicaris, and Gibberella zeae.

In a preferred embodiment, the filamentous fungal parent cell is anAspergillus cell. In another preferred embodiment, the filamentousfungal parent cell is an Acremonium cell. In another preferredembodiment, the filamentous fungal parent cell is a Fusarium cell, e.g.,a Fusarium cell of the section Elegans or of the section Discolor. Inanother preferred embodiment, the filamentous fungal parent cell is aHumicola cell. In another preferred embodiment, the filamentous fungalparent cell is a Myceliophthora cell. In another preferred embodiment,the filamentous fungal parent cell is a Mucor cell. In another preferredembodiment, the filamentous fungal parent cell is a Neurospora cell. Inanother preferred embodiment, the filamentous fungal parent cell is aPenicillium cell. In another preferred embodiment, the filamentousfungal parent cell is a Thielavia cell. In another preferred embodiment,the filamentous fungal parent cell is a Tolypocladium cell. In anotherpreferred embodiment, the filamentous fungal parent cell is aTrichoderma cell. In a more preferred embodiment, the filamentous fungalparent cell is an Aspergillus oryzae, Aspergillus niger, Aspergillusfoetidus, Aspergillus nidulans, or Aspergillus japonicus cell. Inanother more preferred embodiment, the filamentous fungal parent cell isa Fusarium strain of the section Discolor (also known as sectionFusarium). For example, the filamentous fungal parent cell may be aFusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium reticulatum, Fusarium roseum,Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, orFusarium trichothecioides cell. In another preferred embodiment, thefilamentous fungal parent cell is a Fusarium strain of the sectionElegans, e.g., Fusarium oxysporum. In another more preferred embodiment,the filamentous fungal parent cell is a Humicola insolens or Humicolalanuginosa cell. In another more preferred embodiment, the filamentousfungal parent cell is a Myceliophthora thermophilum cell. In anothermore preferred embodiment, the filamentous fungal parent cell is a Mucormiehei cell. In another more preferred embodiment, the filamentousfungal parent cell is a Neurospora crassa cell. In another morepreferred embodiment, the filamentous fungal parent cell is aPenicillium purpurogenum cell. In another more preferred embodiment, thefilamentous fungal parent cell is a Thielavia terrestris cell. Inanother more preferred embodiment, the Trichoderma cell is a Trichodermareesei, Trichoderma viride, Trichoderma longibrachiatum, Trichodermaharzianum, or Trichoderma koningii cell.

The mutant cells produced in the first step are then screened for thosemutant cells which (a) exhibit a more restricted colonial phenotypeand/or more extensive hyphal branching than the parent cell; and (b)have an improved property for production of a heterologous polypeptidethan the parent cell, when the mutant and parent cells are culturedunder the same conditions. In a preferred embodiment, the mutant cellsare inspected first for the more restricted colonial phenotype and/ormore extensive hyphal branching, more preferably, first for the morerestricted colonial phenotype followed by the more extensive hyphalbranching.

The mutant cells of the present invention may have a colonial phenotypewhich is more restricted than the parent cell when the mutant and parentcells are grown on the same solid medium. A mutant cell having “morerestricted colonial phenotype” is defined herein as a mutant cell havinga reduced radial extension rate than a parent cell when the mutant celland parent cell are grown on the solid medium. Preferably, the colonialphenotype of the mutant cells is at least about 10%, more preferably atleast about 20%, and most preferably at least about 30% more restrictedthan the parent cell.

The mutant cells of the present invention may also have a more extensivehyphal branching than the parent cell. A mutant cell having a “moreextensive hyphal branching” is defined herein as a mutant cell having ahyphal growth unit length which is at least 10% less than the hyphalgrowth unit length of the parent cell. Preferably, the hyphal branchingof the mutant cell is at least about 20% more branched, and morepreferably at least about 30% more branched than the parent cell.Measurement of the hyphal growth unit length may be made according tothe method of Trinci et al., 1973, Archiv für Mikrobiologie 91: 127–136.One way of making this determination is to measure the average distancebetween branches in fungal hyphae (see, for example, Withers et al.,1994, Mycological Research 98: 95–100).

The mutant cells of the present invention also have an improved propertyfor production of a heterologous polypeptide than the parent cell, whenthe mutant and parent cells are cultured under the same conditions. Themutants obtained by the methods of the present invention may possessimproved growth characteristics in fermentation where the morphologygives rise to lower viscosity in the fermenter, in turn leading toeasier mixing, better aeration, better growth, and ultimately, enhancedyield of heterologous polypeptide produced by the mutant strain relativeto the parent strain. In a preferred embodiment, the improved propertyis selected from the group consisting of(a) increased yield of theheterologous polypeptide, (b) improved growth, (c) lower viscosity, and(d) better secretion. In a most preferred embodiment, the improvedproperty is increased yield of the heterologous polypeptide. In anothermost preferred embodiment, the improved property is improved growth. Inanother most preferred embodiment, the improved property is lowerviscosity. In another most preferred embodiment, the improved propertyis better secretion.

In order to determine whether a mutant cell has an improved property forproduction of a heterologous polypeptide than the parent cell, a nucleicacid construct comprising a nucleic acid sequence encoding theheterologous polypeptide of interest is introduced into both the parentstrain and the morphological mutant, e.g., by transformation. “Nucleicacid construct” is defined herein as a nucleic acid molecule, eithersingle- or double-stranded, which is isolated from a naturally occurringgene or which has been modified to contain segments of nucleic acidwhich are combined and juxtaposed in a manner which would not otherwiseexist in nature. The mutant cell is preferably transformed with a vectorcomprising the nucleic acid construct followed by integration of thevector into the host chromosome. “Transformation” means introducing anucleic acid construct into a host cell so that the construct ismaintained as a chromosomal integrant. Integration is generallyconsidered to be an advantage as the nucleic acid sequence encoding theheterologous polypeptide is more likely to be stably maintained in thecell. Integration of the vector into the host chromosome occurs byhomologous or non-homologous recombination. Transformation is achievedusing those techniques adapted for the fungal host being used, many ofwhich are well known in the art. Suitable procedures for transformationof Aspergillus cells are described in EP 238 023, Christensen et al.,1988, Bio/Technology 6:1419–1422, and Yelton et al., 1984, Proceedingsof the National Academy of Sciences USA 81:1470–1474. A suitable methodof transforming Fusarium species is described by Malardier et al., 1989,Gene 78:147–156 or in copending U.S. Ser. No. 08/269,449.

After transformation of the mutant and the parent cell with a vectorcontaining a gene encoding a heterologous polypeptide, spores gatheredfrom the mutants and parent are used to inoculate liquid medium. After asuitable period of growth, supernatants are tested for activity of thepolypeptide.

When the improved property is yield, the levels of expression of theheterologous polypeptide are compared between the mutant and parentstrains. In such case, the productive phase of the mutant's fermentationshould be extended. In a preferred embodiment, the morphological mutantproduces at least about 10% more heterologous polypeptide than theparent strain when each strain is cultured under identical conditions.More preferably, the mutant produces at least 20%, and most preferablyat least 30%, more heterologous polypeptide. In some cases, the mutantmay produce as much as 50%–100% more polypeptide, or even higher. Sincein all cultures it is expected that a range of expression levels maybeobserved, it is understood that this figure can represent the mean,median or maximum level of expression in a population of transformantstrains.

When the improved property is reduced viscosity, viscosity can bedetermined by any means known in the art, e.g., Brookfield rotationalviscometry (defined or unlimited shear distance and any type of spindleconfiguration), kinematic viscosity tubes (flow-through tubes), fallingball viscometer or cup-type viscometer. The preferred host cells of thepresent invention exhibit about 90% or less of the viscosity levelproduced by an the parent cell under identical fermentation conditions,preferably about 80% or less, and more preferably about 50% or less.

The present morphological mutants can be used to express any prokaryoticor eukaryotic heterologous peptide or polypeptide of interest, and arepreferably used to express eukaryotic peptides or polypeptides. Ofparticular interest for these species is their use in expression ofheterologous polypeptides, in particular fungal polypeptides, especiallyfungal enzymes. The morphological mutants can be used to express enzymessuch as a hydrolase, an oxidoreductase, an isomerase, a ligase, a lyase,or a transferase. More preferably, the enzyme is an aminopeptidase, anamylase, a carboxypeptidase, a catalase, a cellulase, a chitinase, acutinase, a cyclodextrin glycosyl transferase, a deoxyribonuclease, anesterase, a glucoamylase, an alpha-galactosidase, a beta-galactosidase,an alpha-glucosidase, beta-glucosidase, a haloperoxidase, an invertase,a laccase, a lipase, a mannosidase, a mutanase, an oxidase, apectinolytic enzyme, a peroxidase, a phenoloxidase, phytase, aproteolytic enzyme, a ribonuclease, a xylanase, or a xylose isomerase.It will be understood by those skilled in the art that the term “fungalenzymes” includes not only native fungal enzymes, but also those fungalenzymes which have been modified by amino acid substitutions, deletions,additions, or other modifications which may be made to enhance activity,thermostability, pH tolerance and the like. Other polypeptides that canbe expressed include, but are not limited to, mammalian polypeptidessuch as insulin, insulin variants, receptor proteins and portionsthereof, and antibodies and portions thereof.

The mutants may also be used in recombinant production of polypeptideswhich are native to the host cells. Examples of such use include, butare not limited to, placing a gene encoding the polypeptide under thecontrol of a different promoter to enhance expression of thepolypeptide, to expedite export of a native polypeptide of interestoutside the cell by use of a signal sequence, or to increase the copynumber of a gene encoding the protein normally produced by the subjecthost cells. Thus, the present invention also encompasses, within thescope of the term “heterologous polypeptide”, such recombinantproduction of homologous polypeptides, to the extent that suchexpression involves the use of genetic elements not native to the hostcell, or use of native elements which have been manipulated to functionin a manner not normally seen in the host cell.

In the present invention, the nucleic acid construct is operably linkedto one or more control sequences capable of directing the expression ofthe coding sequence in the mutant cell under conditions compatible withthe control sequences. The term “coding sequence” as defined herein is asequence which is transcribed into mRNA and translated into apolypeptide of the present invention when placed under the control ofthe control sequences. The boundaries of the coding sequence aregenerally determined by a translation start codon ATG at the 5′-terminusand a translation stop codon at the 3′-terminus. A coding sequence caninclude, but is not limited to, DNA, cDNA, and recombinant nucleic acidsequences.

The term “control sequences” is defined herein to include all componentswhich are necessary or advantageous for expression of the codingsequence of the nucleic acid sequence. Each control sequence may benative or foreign to the nucleic acid sequence encoding the polypeptide.Such control sequences include, but are not limited to, a leader, apolyadenylation sequence, a propeptide sequence, a promoter, a signalsequence, and a transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the nucleic acidsequence encoding a polypeptide.

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence which is recognized by a host cell for expression of thenucleic acid sequence. The promoter sequence contains transcription andtranslation control sequences which mediate the expression of thepolypeptide. The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice and may be obtainedfrom genes encoding extracellular or intracellular polypeptides eitherhomologous or heterologous to the host cell. Examples of suitablepromoters for directing the transcription of the nucleic acid constructsof the present invention in a filamentous fungal cell are promotersobtained from the genes encoding Aspergillus oryzae TAKA amylase (asdescribed in U.S. patent application Ser. No. 08/208,092, the contentsof which are incorporated herein by reference), Rhizomucor mieheiaspartic proteinase, Aspergillus niger neutral alpha-amylase,Aspergillus niger acid stable alpha-amylase, Aspergillus niger orAspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase,Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, Fusariumoxysporum trypsin-like protease (as described in U.S. Pat. No.4,288,627, which is incorporated herein by reference), and hybridsthereof. Particularly preferred promoters for use in filamentous fungalcells are the TAKA amylase, NA2-tpi (a hybrid of the promoters from thegenes encoding Aspergillus niger neutral α-amylase and Aspergillusoryzae triose phosphate isomerase), and glaA promoters.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention. Preferred terminators for filamentous fungalcells are obtained from the genes encoding Aspergillus oryzae TAKAamylase, Aspergillus niger glucoamylase, Aspergillus nidulansanthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusariumoxysporum trypsin-like protease.

The control sequence may also be a suitable leader sequence, anontranslated region of a mRNA which is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleic acid sequence encoding the polypeptide. Any leader sequencewhich is functional in the host cell of choice may be used in thepresent invention. Preferred leaders for filamentous fungal host cellsare obtained from the genes encoding Aspergillus oryzae TAKA amylase andAspergillus oryzae triose phosphate isomerase.

The control sequence may also be a polyadenylation sequence, a sequencewhich is operably linked to the 3′ terminus of the nucleic acid sequenceand which, when transcribed, is recognized by the host cell as a signalto add polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention. Preferred polyadenylation sequences forfilamentous fungal host cells are obtained from the genes encodingAspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase,Aspergillus nidulans anthranilate synthase, and Aspergillus nigeralpha-glucosidase.

The control sequence may also be a signal peptide coding region, whichcodes for an amino acid sequence linked to the amino terminus of thepolypeptide which can direct the expressed polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to that portion of the coding sequence which encodes thesecreted polypeptide. The foreign signal peptide coding region may berequired where the coding sequence does not normally contain a signalpeptide coding region. Alternatively, the foreign signal peptide codingregion may simply replace the natural signal peptide coding region inorder to obtain enhanced secretion of the polypeptide relative to thenatural signal peptide coding region normally associated with the codingsequence. The signal peptide coding region may be obtained from aglucoamylase or an amylase gene from an Aspergillus species, a lipase orproteinase gene from a Rhizomucor species, the gene for the alpha-factorfrom Saccharomyces cerevisiae, an amylase or a protease gene from aBacillus species, or the calf preprochymosin gene. However, any signalpeptide coding region capable of directing the expressed polypeptideinto the secretory pathway of a host cell of choice may be used in thepresent invention. An effective signal peptide coding region forfilamentous fungal host cells is the signal peptide coding regionobtained from Aspergillus oryzae TAKA amylase gene, Aspergillus nigerneutral amylase gene, the Rhizomucor miehei aspartic proteinase gene,the Humicola lanuginosa cellulase gene, or the Rhizomucor miehei lipasegene.

The control sequence may also be a propeptide coding region, which codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to mature active polypeptide bycatalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from theBacillus subtilis alkaline protease gene (aprE), the Bacillus subtilisneutral protease gene (nprT), the Saccharomyces cerevisiae alpha-factorgene, or the Myceliophthora thermophilum laccase gene (WO 95/33836).

The vector may be any vector which can be conveniently subjected torecombinant DNA procedures and can bring about the expression of thenucleic acid sequence encoding the polyppetide. The choice of the vectorwill typically depend on the compatibility of the vector with the hostcell into which the vector is to be introduced. The vectors may belinear or closed circular plasmids. The vector system may be a singlevector or plasmid or two or more vectors or plasmids which togethercontain the total DNA to be introduced into the genome of the host cell.The vectors of the present invention preferably contain an element(s)that permits stable integration of the vector into the host cell genome.For integration, the vector may rely on the nucleic acid sequenceencoding the polypeptide or any other element of the vector for stableintegration of the vector into the genome by homologous or nonhomologousrecombination. Alternatively, the vector may contain additional nucleicacid sequences for directing integration by homologous recombinationinto the genome of the host cell. The additional nucleic acid sequencesenable the vector to be integrated into the host cell genome at aprecise location(s) in the chromosome(s). To increase the likelihood ofintegration at a precise location, the integrational elements shouldpreferably contain a sufficient number of nucleic acids, such as 100 to1,500 base pairs, preferably 400 to 1,500 base pairs, and mostpreferably 800 to 1,500 base pairs, which are highly homologous with thecorresponding target sequence to enhance the probability of homologousrecombination. The integrational elements may be any sequence that ishomologous with the target sequence in the genome of the host cell.Furthermore, the integrational elements may be non-encoding or encodingnucleic acid sequences. On the other hand, the vector may be integratedinto the genome of the host cell by non-homologous recombination.

The vectors preferably contain one or more selectable markers whichpermit easy selection of transformed cells. A selectable marker is agene the product of which provides for biocide resistance, resistance toheavy metals, prototrophy to auxotrophs, and the like. A selectablemarker for use in a filamentous fungal host cell may be selected fromthe group including, but not limited to, amdS(acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), trpC (anthranilate synthase), and glufosinateresistance markers, as well as equivalents from other species. Preferredfor use in an Aspergillus cell are the amdS and pyrG markers ofAspergillus nidulans or Aspergillus oryzae and the bar marker ofStreptomyces hygroscopicus. Furthermore, selection may be accomplishedby co-transformation, e.g., as described in WO 91/17243, where theselectable marker is on a separate vector.

According to a preferred embodiment of the present invention, the hostis transformed with a single DNA vector including both the selectionmarker and the remaining heterologous DNA to be introduced, includingpromoter, the gene for the desired polypeptide and transcriptionterminator and polyadenylation sequences.

The procedures used to ligate the elements described above to constructthe nucleic acid constructs and vectors are well known to one skilled inthe art (see, e.g., Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,1989, supra).

The present invention also relates to methods of producing aheterologous polypeptide, comprising: (a) cultivating a mutant cell ofthe present invention which comprises a nucleic acid sequence encodingthe heterologous polypeptide; and (b) recovering the heterologouspolypeptide.

The cells are cultivated in a nutrient medium suitable for production ofthe polypeptide using methods known in the art. For example, the cellmay be cultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art (see, e.g., references forbacteria and yeast; Bennett, J. W. and LaSure, L., editors, More GeneManipulations in Fungi, Academic Press, CA, 1991). Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it is recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide. Procedures for determiningenzyme activity are well known in the art.

The resulting polypeptide may be recovered by methods known in the art.For example, the polypeptide may be recovered from the nutrient mediumby conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. The recovered polypeptide may then be further purified bya variety of chromatographic procedures, e.g., ion exchangechromatography, gel filtration chromatography, affinity chromatography,or the like.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), or extraction (see, e.g., ProteinPurification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, NewYork, 1989).

EXAMPLES

Strains and Media

The starting strains are alpha-amylase deficient, pyrG-negativeAspergillus oryzae HowB425 and Fusarium A3/5. Morphological mutants ofFusarium A3/5 designated CC1-3, CC2-3, and MC3-5 (Wiebe et al., 1992,Mycological Research 96: 555–562; Wiebe et al., 1991, MycologicalResearch 95: 1284–1288; Wiebe et al., 1991, Mycological Research 96:555–562) are highly branched, colonial variants.

PDA plates contain 39 g/l Potato Dextrose Agar (Difco) and aresupplemented with 10 mM uridine for pyrG auxotrophs unless otherwiseindicated.

MY50N medium is comprised of 62.5 g of Nutriose, 2.0 g of MgSO₄-7H₂O,2.0 g of KH₂PO₄, 4.0 g of citric acid, 8.0 g of yeast extract, 2.0 g ofurea, 0.1 g of CaCl₂, and 0.5 ml of trace metals solution pH 6.0 perliter. MY50N shake-flask medium is diluted 1:100 with glass distilledwater for use in microtiter growth experiments (MY50N/100). Cultures aregrown at a temperature between 28–37° C.

Minimal medium plates are comprised of 6.0 g of NaNO₃, 0.52 g of KCl,1.52 g of KH₂PO₄, 1.0 ml of trace metals solution, 20 g of Nobel Agar(Difco), 20 ml of 50% glucose, 20 ml of methionine (50 g/l), 20 ml ofbiotin (200 mg/l), 2.5 ml of 20% MgSO₄-7H₂O, and 1.0 ml of streptomycinper liter. The agar medium is adjusted to pH 6.5 prior to autoclavingand then glucose, methionine, biotin, MgSO₄-7H₂O, and streptomycin areadded as sterile solutions to the cooled autoclaved medium and pouredinto plates.

The trace metals solution (1000×) is comprised of 22 g of ZnSO₄-7H₂O, 11g of H₃BO₃, 5 g of MnCl₂-4H₂O, 5 g of FeSO₄-7H₂O, 1.6 g of CoCl₂-5H₂O,1.6 g of (NH₄)₆Mo₇O₂₄, and 50 g of Na₄EDTA per liter.

COVE plates are comprised of 343.3 g of sucrose, 20 ml of COVE saltssolution, 10 ml of 1 M acetamide, 10 ml of 3 M CsCl, and 25 g of Nobelagar per liter. The COVE salts (50×) solution is comprised of 26 g ofKCl, 26 g of MgSO₄-7H₂O, 76 g of KH₂PO₄, and 50 ml of COVE trace metalssolution. COVE trace metals solution is comprised of 0.04 g ofNaB₄O₇-10H₂O, 0.040 g of CuSO₄-5H₂O, 0.70 g of FeSO₄—H₂O, 0.80 g ofNa₂MoO₂-2H₂O, and 10 g of ZnSO₄ per liter.

M400Da medium is comprised of 50 g of maltodextrin, 2.0 g of MgSO₄-7H₂O,2.0 g of KH₂PO₄, 4.0 g of citric acid, 8.0 g of yeast extract, 2.0 g ofurea, and 0.5 ml of trace metals solution per liter. The medium isadjusted to pH 6.0 with 5 N NaOH. The trace metals solution is comprisedof 14.3 g of ZnSO₄-7H₂O, 2.5 g of CuSO₄-5H₂O, 0.5 g of NiCl₂-6H₂O, 13.8g of FeSo₄-7H₂O, 8.5 g of MnSO₄—H₂O, and 3.0 g of citric acid per liter.

Example 1 Mutagenesis of Aspergillus oryzae strain HowB425

Aspergillus oryzae strain HowB425 spores are harvested from solid mediumand suspended to a concentration of 2.2×10⁷/ml in 0.01% Tween 80. Fiveml of spore suspension are pipetted into a 90 mm plastic petri dish andthe spores are irradiated for one minute with ultraviolet light toapproximately 5% survival. The mutagenized spores are kept in the darkfor one hour and then plated to PDA+50 mg/l uridine plates.

The frequencies of spore color mutants obtained from this mutagenesistreatment are 8.8×10⁻⁵ for white and 5.9×10⁻⁵ for yellow spored mutants.A total of about 34,000 viable colonies (250 to 800 per plate) arescreened visually for a restricted colonial phenotype. Eighty-eightrestricted colonials are selected. The edges of the restricted coloniesgrowing on the plate are examined under a microscope (200×) for a highmycelial branching phenotype. Thirty-six of the selected colonials areselected as having a more extensive hyphal branching pattern than theAspergillus oryzae HowB425 control strain and are purified byrestreaking spores onto PDA+uridine plates. After growth andsporulation, the strains are repurified in a similar fashion. The 36mutants are re-examined for the colonial and high-branching phenotypes.Twelve of the 36 retested positive in both assays and are selected forheterologous polypeptide expression analysis. The frequency of mutantsrecovered is 12/34,000 or about 3.5×10⁻⁴. The colonial mutants areclassified by examination of their hyphal branching phenotypes (TableI). The colony morphology of the control strain and one of the mutantson PDA+uridine solid medium are shown in FIG. 2.

TABLE I Phenotypes of Morphological Mutants Phenotypes Strains Wild Typegrowth and low branching HowB425 (control) Colonial growth and mediumbranching JeRS306 JeRS307 JeRS314 JeRS316 JeRS318 Colonial growth andhigh branching JeRS303 JeRS304 JeRS315 JeRS320 Colonial growth and veryhigh branching JeRS313 Colonial growth and highly branched short hyphaeJeRS317 Colonial growth and highly branched very short hyphae JeRS305

Example 2 Lipase Expression Plasmid

A map of the lipase expression plasmid pJeRS23 is shown in FIG. 1.pJeRS23 contains the amdS gene from Aspergillus nidulans from bases −118to 2191 (relative to the ATG start codon), the pTAKA-TPI/Lipolase/AMGtlipase expression cassette from pMHan37, the Aspergillus oryzae pyrGgene, and pUC19 sequences.

Example 3 Aspergillus oryzae Transformation

Cultures to be transformed are grown in 20 ml of 1% yeast extract-2%Peptone (Difco)-2.5% glucose at 37° C. for 16–20 hours with agitation.Each culture is mixed with 10 ml of 1.2 M MgSO₄, and the mycelia arerecovered by filtration on Miracloth (CalBiochem, La Jolla, Calif.) orby centrifugation, washed with 1.2 M MgSO₄, and then resuspended in 10ml of 5 mg/ml NOVOZYM 234 (Novo Nordisk A/S, Bagsvaerd, Denmark) in 1.2M MgSO₄. The suspension is incubated with gentle agitation forapproximately one hour at 37° C. to generate protoplasts. Undigestedmycelia are removed by filtration through a layer of sterile Miracloth.Protoplasts are recovered by centrifugation at 3600×g. They are thenwashed with 10 ml of ST (1 M sorbitol-10 mM Tris pH 7.5), centrifuged,washed with 10 ml of STC (1 M sorbitol-10 mM Tris pH 7.5–10 mM CaCl₂),centrifuged, and then resuspended in 1.0 ml of STC. The concentration ofprotoplasts is determined and the final concentration is adjusted tobetween 2×10⁶ and 1×10⁷/ml with STC. An aliquot of 0.1 ml protoplasts ismixed with 5 μl of pJeRS23 DNA (about 5 μg) in a Falcon 2059polypropylene tube and incubated at room temperature for 20 minutes. Oneml of SPTC (0.8 M sorbitol-40% polyethylene glycol 4000-50 mM CaCl₂-50mM Tris pH 8) is added and the suspension is mixed with gentle shaking.The suspension is incubated at room temperature for 20 minutes and then7 ml of molten overlay agar (1× COVE salts, 0.8 M sucrose, 1% low meltagarose) is added and the suspension is poured onto a COVE plate. Theplates are incubated at 37° C.

Example 4 Lipase Assay

Assay substrate is prepared by diluting 1:5 the stock substrate (10 μlof p-nitrophenylbutyrate/ml DMSO) into MC buffer (3 mM CaCl₂-0.1M MOPSpH 7.5) immediately before use. Standard Lipolase® contains 1000 LU/mlof 50% glycerol-0.66 mM CaCl₂-33 mM Tris pH 7.5 and is stored at −20° C.Standard Lipolase® is diluted 1/100 in MC buffer just before use. Brothsamples are diluted in MC buffer and 100 μl aliquots of the dilutedbroth samples are pipetted into 96-well microtiter dishes followed by100 μl of diluted substrate. The absorbance at nm is recorded as afunction of time. Broth lipase units/ml (LU/ml) are calculated relativeto a Lipolase® standard.

Example 5 Lipolase® Expression

Each of the twelve mutants is transformed with the Lipolase® expressionplasmid pJeRS23 described in Example 2 and the transformants areselected by their prototrophy for uridine and ability to grow usingacetamide as sole nitrogen source. A parallel transformation isperformed with the parent strain Aspergillus oryzae HowB425. Theconidiated transformants are restreaked once to COVE plates and sporesfrom individual colonies are used to inoculate a 90 mm COVE plate. Aftersporulation, the spores are harvested in 0.01% Tween 80. A 10 μl aliquotof each spore suspension is used to inoculate a well in a 24-wellmicrotiter plate that contains 1 ml of MY50N/100 liquid medium.Experiments are started on two different days (Experiments A and B) withthe entire set of Aspergillus oryzae HowB425 control transformantsincluded each day. The microtiter plates are grown for 3–5 days at 37°C., 100 rpm agitation, and the culture supernatants are assayed forlipase activity as described in Example 4.

The results are shown in Table II. A graphic representation of two ofthe mutants is shown in FIGS. 3 and 4. Although it is typical thatindividual transformants obtained following the introduction of DNAexpression plasmids into any given Aspergillus oryzae host will vary intheir ability to produce and secrete a heterologous polypeptide, and thenumber of transformants in each mutant strain is fairly small, theexpression profiles for mutants JeRS316 (FIG. 3) and JeRS317 (FIG. 4)appear to be shifted further to higher lipase expression values ascompared with the control.

TABLE II Lipolase ® Expression in Morphological Mutants Standard ofnumber of Mean Deviation Median Max Strain transformants (LU/ml) (LU/ml)(LU/ml) (LU/ml) Experiment A HowB425 12 6.49 3.28 5.47 12.10 JeRS305 82.11 2.64 0.62 6.61 JeRS306 12 7.44 5.49 6.57 15.40 JeRS307 10 8.31 6.496.74 24.00 JeRS313 5 4.54 3.84 2.40 8.98 JeRS315 6 8.96 8.60 7.27 25.80JeRS317 9 12.20 6.85 10.97 28.00 JeRS318 9 1.53 3.68 0.00 11.20 JeRS3209 9.47 7.97 5.11 22.56 Experiment B HowB425 12 12.10 4.04 11.40 20.00JeRS303 12 7.51 9.55 4.34 31.70 JeRS304 10 12.90 11.30 9.60 36.50JeRS314 12 11.40 5.89 12.20 21.60 JeRS316 12 18.00 15.00 13.40 44.50

Example 6 Fermentation of Aspergillus oryzae mutant JeRS316

To determine if the morphology mutants exhibit a superior fermentationbehavior in comparison with the parent wild type morphology strain, onetransformant each of the parent strain Aspergillus oryzae HowB425 andthe mutant strain JeRS316 transformed with plasmid pJeRS23 are grown ina tank fermenter under fermentation conditions.

The morphology of the control culture at the end of the fermentation istypical for Aspergillus oryzae grown under these conditions. The cultureis very viscous with a thick and grainy slow mixing appearance. Largeair bubbles are visible in the tank. In contrast, the JeRS316transformant displays a low viscosity, filamentous, easy mixingmorphology with a small degree of pellet formation throughout thefermentation. Large air bubbles are not routinely observed in the tank.The expression of the heterologous lipase is examined and the resultsare shown in FIG. 5. If the mutant is superior to the parent infermentation, the expectation is that the productive phase of thefermentation would be extended for the mutant. The results are reportedas the ratio of [lipase titer in the culture broth at time (x)]/[lipasetiter at time (42 hours)]. This analysis normalizes the expression datafor the fact that not all transformants are equivalent in their absolutelevel of expression. As predicted for an improved morphology mutant, theheterologous polypeptide productive phase of the fermentation isextended significantly in strain JeRS316 as compared with the control.The final expression of the lipase in the broth of the morphology mutantculture is about five times higher in titer than the control.

Example 7 Construction of Fusarium expression Vector pJRoy30

The EcoRV site at −15 in the Fusarium oxysporum trypsin gene promoterofpJRoy20 (Royer et al., 1995, Bio/Technology 13: 1479–1483) and theNcoI site present at +243 in the CAREZYME™ (Novo Nordisk A/S, Bagsværd,Denmark) cellulase coding region are utilized to create an exact fusionbetween the Fusarium oxysporum trypsin gene promoter and the CAREZYME™cellulase gene. A PCR fragment containing −18 to −1 of the Fusariumoxysporum trypsin gene promoter directly followed by −1 to +294 of theCAREZYME™ cellulase gene is generated from the CAREZYME™ vector pCaHj418(see FIG. 6) using the following primers:

FORWARD EcoRV 5′ ctcttggatatctatctcttcaccATGCGTTCCTCCCCCCTCCT 3′ (SEQ IDNO: 1) REVERSE 5′ CAATAGAGGTGGCAGCAAAA 3′ (SEQ ID NO: 2)Lower case letters in the forward primer are bp −24 to −1 of theFusarium oxysporum trypsin gene promoter, while upper case letters arebp 1 to 20 of CAREZYME™.

The PCR conditions used are 95° C. for 5 minutes followed by 30 cycleseach at 95° C. for 30 seconds, 50° C. for 1 minute, and 72° C. for 1minute. The resulting 0.32 kb fragment is cloned into vector pCRII usingInvitrogen's TA Cloning Kit (Invitrogen, La Jolla, Calif.) resulting inpDM148 (see FIG. 7). The 0.26 kb EcoRV/NcoI fragment is isolated frompDM148 and ligated to the 0.69 kb NcoI/BglII fragment from pCaHj418 andcloned into EcoRV/BamHI digested pJRoy20 to create pDM149 (see FIG. 8).

pMT1612 is constructed by introducing a 575 bpBamH1 BamH1 fragmentcontaining the bar gene from pBIT (Straubinger et al., 1992, FungalGenetics Newsletter 39:82–83) into pIC19H (Marsh et al., 1984, Gene32:481–485) cut with BamHI/BglII. The bar gene is then isolated as aBamH1-XhoI fragment and inserted into BamHI-XhoI cut pJaL154 (FIG. 9) togenerate pMT1612 (FIG. 10). The 3.2 kb EcoR1 CAREZYME™ cellulaseexpression cassette is transferred from pDM149 into EcoR1 cut bastamarker pMT1612 to generate pJRoy30 (FIG. 11).

Example 8 Transformation of Fusarium

Fusarium strain A3/5 (ATCC 20334) and Fusarium strain A3/5 highlybranched morphological mutants CC1-3, CC1-8, CC2-3, and MC3-5 (Wiebe etal., 1992, Mycological Research 96:555–562) are grown on 10×15 mm petriplates of Vogels medium (Vogel, 1964, Am. Nature 98:435–446) plus 1.5%glucose and agar for 3 weeks at 25° C. Conidia (approximately 10⁸ perplate) are dislodged in 10 ml of sterile water using a transfer loop andpurified by filtration through 4 layers of cheesecloth and finallythrough one layer of Miracloth. Conidial suspensions are concentrated bycentrifugation. Fifty ml of YPG medium comprised of 1% yeast extract, 2%bactopeptone, and 2% glucose are inoculated with 10⁸ conidia, andincubated for 14 hours at 24° C., 150 rpm. Resulting hyphae are trappedon a sterile 0.4 μm filter and washed successively with steriledistilled water and 1.0 M MgSO₄. The hyphae are resuspended in 10 ml ofNOVOZYM 234™ solution (2–10 mg/ml in 1.0 M MgSO₄) and digested for 15–30minutes at 34° C. with agitation at 80 rpm. Undigested hyphal materialis removed from the resulting protoplast suspension by successivefiltration through 4 layers of cheesecloth and through Miracloth. Twentyml of 1 M sorbitol are passed through the cheesecloth and Miracloth andcombined with the protoplast solution. After mixing, protoplasts(approximately 5×10⁸) are pelleted by centrifugation and washedsuccessively by resuspension and centrifugation in 20 ml of 1 M sorbitoland in 20 ml of STC. The washed protoplasts are resuspended in 4 partsSTC and 1 part SPTC at a concentration of 1–2×10⁸/ml. One hundred μl ofprotoplast suspension are added to 5 μg pJRoy30 and 5 μl heparin (5mg/ml in STC) in polypropylene tubes (17×100 mm) and incubated on icefor 30 minutes. One ml of SPTC is mixed gently into the protoplastsuspension and incubation is continued at room temperature for 20minutes. Twenty five ml of molten solution (cooled to 40° C.) consistingof COVE salts, 25 mM NaNO₃, 0.8 M sucrose and 1% low melting agarose(Sigma Chemical Company, St. Louis, Mo.) are mixed with the protoplastsand then plated onto an empty 150 mm petri plate. Incubation iscontinued at room temperature for 10 to 14 days. After incubation atroom temperature for 24 hours, 25 ml of the identical medium plus basta(5 mg/ml) are overlayed onto the petri plate. Basta is obtained fromAgrEvo (Hoechst Schering, Rodovre, Denmark) and is extracted twice withphenol:chloroform:isoamyl alcohol (25:24:1), and once withchloroform:isoamyl alcohol (24:1) before use.

Example 9 Expression of Cellulase Activity

Transformants of Fusarium A3/5, CC1-3, CC2-3, and MC3-5 are cultured onM400 Da medium in microtiter plates and shake flasks for 7 days at 37°C. One transformant each of Fusarium A3/5, CC 1-3, CC2-3, and MC3-5 arecultivated in fermentors under suitable fermentation conditions in amedium containing typical carbon and nitrogen sources as well as mineralsalts and trace metals.

Cellulase activity is measured using the following procedure. Volumes of5 μl of various dilutions of a cellulase standard and 1–5 μl of samplesare pipetted into a 96-well plate. The cellulase standard (CAREZYME™,Novo Nordisk A/S, Bagsverd, Denmark) is diluted to 150, 100, 50, 25,12.5 and 6.25 ECU/ml in 100 mM MOPS pH 7.0. The substrate is prepared bydissolving azo-carboxymethylcellulose (Azo-CMC) at 2% w/v in 100 mM MOPSpH 7.0 and stirring at 80° C. for 10 minutes. A volume of 65 microlitersof the azo-CMC substrate solution is pipetted into each of the samplewells and mixed. The 96-well plate is incubated in a water bath at 45°C. for 30 minutes and is then placed on ice for 2 minutes. A volume of175 microliters of stop reagent is added to each well and mixed well.The stop reagent is prepared by suspending 0.2 g of ZnCl₂ in 20 ml of0.25 M MOPS pH 7.0 and adding the suspension to 80 ml of acidifiedethanol containing 1.1 ml of concentrated HCl per liter of ethanol. The96-well plate is centrifuged at 3000 rpm for 10 minutes in a Sorval RT6000B centrifuge. After centrifugation is complete, 50 μl of eachsupernatant is transferred to a new 96-well plate containing 50 μl ofwater per well. The absorbance at 600 nm is measured.

The results for the microtiter plate, shake flask, and fermentorcultures are presented in Table III where the maximum cellulase yield isnormalized to 1.0. In microtiter plate culture, transformants of CC2-3and MC3-5 produce levels of cellulase which are 22% and 46% higher,respectively, compared to the parent strain. In shake flask culture,transformants of CC 1-3, CC2-3 5, and MC3-5 produce levels of cellulasewhich are 85%, 54%, and 7% higher, respectively, compared to the parentstrain. In fermentors, of CC1-3, CC2-3 5, and MC3-5 produce levels ofcellulase which are 136%, 3%, and 8% higher, respectively, compared tothe parent strain.

TABLE III Production of cellulase by transformants of the wild typestrain (A3/5) and morphological mutants (CC1-3, CC2-3, and MC3-5).Microtiter Plate Shake Flask Fermentor Host Maximum° n* Maximum nMaximum n A3/5 1.0  5 1.0  6 1.0  1 CC1-3 0.29 4 1.85 2 2.36 1 CC2-31.22 1 1.54 1 1.03 1 MC3-5 1.46 2 1.07 2 1.08 1 °The maximum cellulaseyield of the Fusarium A3/5 parent strain is normalized to 1.0 for thehighest activity observed under each set of growth conditions. *nrepresents the number of transformants analyzed.

1. A method for obtaining a mutant cell which produces a heterologousprotein, comprising: (a) producing a first population of presumptivemutant cells from a Humicola, Mucor, Myceliophthora, Scytalidium,Thielavia, or Tolypocladium parent cell; (b) identifying from the firstpopulation a second population of presumptive mutant cells having a morerestricted colonial phenotype or a more extensive hyphal branching thanthe parent cell; and (c) identifying from the second population themutant cell, comprising a nucleic acid sequence encoding a heterologouspolypeptide, which has a radial extension rate which is at least 10%less than the parent cell, has a hyphal growth unit length that is atleast 10% less than the parent cell, and has one or more improvedproperties selected from the group consisting of (i) produces at leastabout 10% more heterologous polypeptide than the parent cell, (ii)exhibits about 90% or less of the viscosity of the parent cell, and(iii) secretes more heterologous protein than the parent cell, whencultivated under the same conditions; and (d) obtaining the mutant cell.2. The method of claim 1, wherein in step (b), the second population ofpresumptive mutant cells has a more restricted colonial phenotype and amore extensive hyphal branching than the parent cell.
 3. The method ofclaim 1, wherein the radial extension rate is at least 20% less than theparent cell.
 4. The method of claim 1, wherein the hyphal growth unitlength is at least 20% less than the parent cell.
 5. The method of claim1, wherein the mutant cell produces at least about 10% more heterologouspolypeptide than the parent cell.
 6. The method of claim 1, wherein themutant cell exhibits about 90% or less of the viscosity of the parentcell.
 7. The method of claim 1, wherein the mutant cell has a furtherimproved property of an improved growth.
 8. A method for producing aheterologous polypeptide, comprising: (a) cultivating a mutant cell of aHumicola, Mucor, Myceliophthora, Scytalidium, Thielavia, orTolypocladium parent cell under conditions suitable for production ofthe heterologous polypeptide, wherein the mutant cell comprises anucleic acid sequence encoding the heterologous polypeptide, and whereinthe mutant cell has a radial extension rate which is at least 10% lessthan the parent cell, has a hyphal growth unit length that is at least10% less than the parent cell, and has one or more improved propertiesselected from the group consisting of (i) produces at least about 10%more heterologous polypeptide than the parent cell, (ii) exhibits about90% or less of the viscosity of the parent cell, and (iii) secretes moreheterologous protein than the parent cell, when cultivated under thesame conditions; and (b) recovering the heterologous polypeptide.
 9. Themethod of claim 8, wherein the radial extension rate is at least 20%less than the parent cell.
 10. The method of claim 8, wherein the hyphalgrowth unit length is at least 20% less than the parent cell.
 11. Themethod of claim 8, wherein the mutant cell produces at least about 10%more heterologous polypeptide than the parent cell.
 12. The method ofclaim 8, wherein the mutant cell exhibits about 90% or less of theviscosity of the parent cell.
 13. The method of claim 8, wherein themutant cell has a further improved property of an improved growth.
 14. Amutant cell of a Humicola, Mucor, Myceliophthora, Scytalidium,Thielavia, or Tolypocladium parent cell comprising a nucleic acidsequence encoding a heterologous polypeptide, wherein the mutant cellhas a radial extension rate which is at least 10% less than the parentcell, has a hyphal growth unit length that is at least 10% less than theparent cell, and has one or more improved properties selected from thegroup consisting of (i) produces at least about 10% more heterologouspolypeptide than the parent cell, (ii) exhibits about 90% or less of theviscosity of the parent cell, and (iii) secretes more heterologousprotein than the parent cell, when cultivated under the same conditions.15. The mutant cell of claim 14, wherein the radial extension rate is atleast 20% less than the parent cell.
 16. The mutant cell of claim 14,wherein the hyphal growth unit length is at least 20% less than theparent cell.
 17. The mutant cell of claim 14, wherein the mutant cellproduces at least about 10% more heterologous polypeptide than theparent cell.
 18. The mutant cell of claim 14, wherein the mutant cellexhibits about 90% or less of the viscosity of the parent cell.
 19. Themethod of claim 14, wherein the mutant cell has a further improvedproperty of an improved growth.